Blood clotting or hemostasis is an important protective mechanism of the body for sealing wounds caused from injury to the body. Hemostasis takes place in two phases. Primary (cellular) hemostasis serves to quickly stop bleeding and minimize blood loss. Primary hemostasis involves injured cells of the endothelium and the underlying layer of cells emitting signals that enable blood platelets (thrombocytes) to accumulate in a region of an injured blood vessel, forming a plug that provisionally seals the wound. Secondary (plasmatic) hemostasis or coagulation is initiated at the same time as primary hemostasis and involves a process by which blood clots. More specifically, coagulation is controlled by a signaling coagulation cascade consisting of thirteen coagulation factors that interact and activate each other. At the end of the coagulation cascade, fibrinogen is converted into fibrin. A network of fibrin fibers reinforces wound closure, and platelets and other blood cells get caught in this network and form a blood clot (thrombus). Lastly, platelets and the endothelium release growth factors that control a wound-healing process. At the end of these processes, the fibrin network is dissolved by enzymes in the blood plasma.
The coagulation cascade of secondary hemostasis is based on catalytic conversion of fibrinogen, a soluble plasma protein, to insoluble fibrin. The enzyme catalyzing this reaction is thrombin, which does not permanently circulate in the blood in an active form but exists as prothrombin, the inactive precursor of thrombin. The coagulation cascade leading to active thrombin consists of two pathways, the extrinsic and the intrinsic pathways, which converge into a common pathway that includes active thrombin catalyzing the conversion of fibrinogen to fibrin. The extrinsic pathway is initiated at the site of injury in response to the release of tissue factor (factor III) and thus, is also known as the tissue factor pathway. Tissue factor is a cofactor in the factor VIIa-catalyzed activation of factor X (inactive) to factor Xa (active). The second, more complex, intrinsic pathway is activated by clotting factors VIII, IX, X, XI, and XII associated with platelets. Also required are the proteins prekallikrein (PK) and high-molecular-weight kininogen (HK or HMWK), as well as calcium ions and phospholipids secreted from platelets. Each of these constituents leads to the conversion of factor X to factor Xa. The common point in both pathways is the activation of factor X to factor Xa. Factor Xa is an enzyme (e.g., a serine endopeptidase) that cleaves prothrombin in two places (an arg-thr and then an arg-ile bond), which yields active thrombin and ultimately results in the conversion of fibrinogen to fibrin.
Consequently, the coagulation cascade is a suitable target for diagnosing and treating diseases involving dysregulated blood clotting or the absence of clotting. For example, the diagnosis of hemorrhagic conditions such as hemophilia, where one or more of the thirteen blood clotting factors involved in the coagulation cascade may be defective, can be achieved by a wide variety of coagulation tests. In addition, several tests have been developed to monitor the progress of thrombolytic therapy. Other tests have been developed to signal a prethronibolytic or hypercoagulable state, or monitor the effect of administering protamine to patients during cardiopulmonary bypass surgery. However, the main value of coagulation tests is in monitoring oral and intravenous anticoagulation therapy. Three of the key diagnostic tests are prothrombin time (PT), activated partial thromboplastin time (aPTT), and activated clotting time (ACT).
Activators of blood clotting via each of the extrinsic and intrinsic pathways are known. For example, activation of the intrinsic pathway can occur by a variety of negatively charged insoluble substances. The action of these substances involves the specific adsorption of factor XII and other proteins of the contact activation system, leading to an acceleration of the proteolytic activation of factor XII, prekallikrein, and factor XI. Although most known activators of the intrinsic pathway such as glass, silica, celite, and kaolin are insoluble, activation has been reported to occur by polyanions heparin, dextran sulfate, carrageenans, and soluble ellagic acid (see, e.g., Girolami et al., 1966, Blood, 27(1):93-102). Unique amongst these substances is ellagic acid, which is a ubiquitous polyphenol found in many fruits, nuts and seeds, and an effective activated cephaloplastin reagent (commercially available cephaloplastin reagents typically also include a phospholipid, typically a cephalin, as a platelet substitute). Unfortunately, the aqueous insolubility of ellagic acid makes it disfavored for use as an activating reagent in many coagulation assays such as the aPTT that are designed for use in single use coagulation assay devices because activating reagents comprising ellagic acid often take a long time to prepare and are not stable after extended periods of storage (e.g., greater than 1-2 days at room temperature).
To address the aqueous insolubility of ellagic acid, a number of solutions have been proposed. For example, ellagic acid has been shown to be soluble in aprotic polar solvents such as N,N-diemthyl formamide, gamma butyrolactone, acetonitrile, and N-methyl-2-pyrrolidone (NMP) (see, e.g., Reitze et al., 2001, Holzforschung, 55:171-5). These solvents, however, are either toxic, not compatible with coagulation assays, or are not appropriate for use in a single use coagulation assay device as they pose safety, shelf-life, and other problems. Alternatively, soluble salts of ellagic acid have been generated by adding sodium hydroxide (see, e.g., U.S. Pat. No. 3,486,981). The soluble salt preparations, however, generate derivatives of ellagic acid, not solubilized ellagic acid, making their use in a single use coagulation assay device less desirable. Additionally, ellagic acid preparations with sodium hydroxide tend to be unstable for longer period of times, and thus, a preservative such as phenol is typically added to the ellagic acid preparation to stabilize the solution for longer periods of time (see, e.g., U.S. Pat. No. 5,055,412). These solutions, however, often take a long time to prepare, a manufacturing drawback which increases expense. Further, phenol is corrosive and causes liver and kidney damage, is a mutagen and potential reproductive hazard.
Consequently, many of the above-mentioned solutions to solubilize ellagic acid are not amenable for the preparation of ellagic acid as a reagent in single use coagulation assay devices. For example, many of the organic solvents are toxic, and will not be useful for long term room temperature storage, which is required of most single use disposable cartridges. Moreover, the addition of hydroxides and/or preservatives to solubilize and stabilize ellagic acid generates decomposition products (i.e., an inactive chemical species derived from ellagic acid) based on the dehydration of the alcohol group and/or saponification reaction of the two ester groups found in ellagic acid. Accordingly, the need exists for highly soluble, stable, and manufacturable ellagic acid suspensions for use in a blood-coagulation assay of a single use disposable cartridge.